The integral membrane desaturases are an enzyme family of immense biomedical and industrial importance. The significance of the desaturases arises from their fundamental contributions to lipid compositions and cellular homeostasis. In both eukaryotes and prokaryotes, desaturases produce essential mono- and polyunsaturated precursors to the lipid components of all cell membranes and thus help to control and maintain membrane function. Therefore, desaturases may be involved in human diseases associated with changes in lipid composition, including obesity, diabetes, hypertension, cardiovascular disease, immune disorders, degenerative neurological diseases, and skin diseases. Links between monounsaturated fatty acids and the regulation of apoptosis, neuronal differentiation, and signal transduction have been reported. The influence of monounsaturated fatty acids on apoptosis may be coupled to the development of some tumors (Lu et al., 1997, J. Mol. Carcinog. 20: 204-215; Falvella et al., 2002, Carcinog. 11: 1922-1936). Also, the fatty acid composition of erythrocyte membranes is associated with breast cancer risk (Pala et al., 2001, J. Nat Canc. Inst. 93: 1088-1095).
Stearoyl-CoA desaturase (SCD) catalyzes the rate-determining step in the synthesis of monounsaturated fatty acids. SCD introduces a double bond between positions 9 and 10 of stearoyl-CoA (18:0) and palmitoyl-CoA (16:0). The activity of SCD influences the fatty acid composition of membrane phospholipids, triglycerides, and cholesterol esters. Alterations of SCD activity result in changes of membrane fluidity, lipid metabolism, and metabolic rate.
Transgenic mice (Mus musculus) with a mutation in stearoyl-CoA desaturase 1 (SCD1) have increased energy expenditure, reduced body adiposity, and remain lean when subject to a high calorie diet, despite a higher food intake as compared to control mice (Ntambi et al., 2003, Prog. Lipid. Res. 43: 91-104). These findings, limited to analysis of the SCD function, link SCD function to a major health epidemic, obesity, and identify SCD as potential target for anti-obesity drugs.
Unsaturated fatty acids are also precursors of mycolic acid, a wax-like coating that protects human pathogens such as Mycobacterium tuberculosis from desiccation, macrophage attack, water-soluble antibiotics, and other ameliorative agents. Desaturases are of great importance to insects in the biosynthetic pathways for production of juvenile maturation hormones, and in the use of fatty acids as an energy source during swarming. Desaturases also contribute to the composition of all plant seed oils consumed by humans, and are recognized as relevant enzymes for renewable sources of hydrocarbons.
Each of the above areas involving desaturases has high impact on human health or areas of economic interest. It is therefore important to improve our understanding of the mechanisms in which fatty acid desaturation proteins function, and to understand the consequence of these enzymatic reactions on cellular structure and function.
The desaturase enzyme family is defined by the Pfam database (Bateman et al., 2004, Nucl. Acids Res. 32: D138-D141). SCD from yeast, rat, and mice are each members of the class III diiron family of enzymes. The hallmark of the membrane-bound SCD enzymes is that all contain an eight histidine motif (HX(3-4)H˜ ˜HX(2-3)HH˜ ˜HX(2-3)HH). Site-directed mutagenesis in rat SCD has demonstrated that all eight histidines are essential for activity and it was postulated that at least some of these residues were necessary for binding the iron atoms. Four isoforms of SCD have been identified in mice (SCD1-4). SCD1 is expressed largely in the liver and adipose tissue. SCD2 is expressed in the mouse brain, heart, lungs, kidney, spleen, and adipose tissue. SCD3 is expressed in the skin, Harderian gland, and preputial gland. SCD4 is expressed exclusively in the heart. These mouse isoforms are highly homologous and contain the histidine motif. The physiological roles of these different enzyme isoforms are currently not understood.
Saccharomyces cerevisiae (yeast) contains a single, essential gene (OLE1) that codes for a desaturase enzyme that is homologous to mouse SCDs. A yeast mutant lacking the OLE1 gene is incapable of growing in the absence of unsaturated fatty acids (UFAs). Transformation with an exogenous gene containing desaturase activity would complement an OLE1 deficient mutant.
Mycobacterium tuberculosis contains a single essential gene (DesA3) that codes for a desaturase enzyme that is also homologous to mouse SCDs. Disruption of the DesA3 gene in Mycobacterium is lethal.
DesA3 has been identified as a possible drug target for the treatment of tuberculosis. The development of efficient drugs that can inhibit or destroy the activity of this enzyme can only be accomplished upon understanding of its function. Currently, little is known about DesA3 structure and function. The characterization of DesA3 has been impaired by the lack of understanding of the composition of the enzyme system required for activity. The state of the art involves study of DesA3 alone using impure vesicle preparations obtained from mycobacterial homogenates.
It would be advantageous to identify other essential factors for the function of DesA3 and to then develop a system that will enable the determination of enhanced levels of DesA3 enzymatic activity in vitro. This knowledge could lead to a better understanding of the physiological role of DesA3 and its isoforms in vivo. The generation of such model expression system could also be used for identification of the roles of the proteins involved in a multi-protein composition such as desaturase. The present invention addresses these and other related needs.